大鼠CB1基因真核表达载体的构建及其对CaSki细胞凋亡的影响  

Construction of expression vector containing rat rCB1 gene and its influence on the apoptosis in human cervical cancer CaSki cell line

作　　者：严磊[1] 李晶[2] 赵婷婷[1] 王会娟[1] 王蕾[1] 赖国旗[1,3] 

机构地区：[1]重庆医科大学实验动物中心,重庆400016 [2]重庆三峡医药高等专科学校,重庆404120 [3]重庆市啮齿类实验动物工程技术研究中心,重庆400016

出　　处：《中国实验动物学报》2015年第2期153-158,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：重庆市教委自然科学基金项目(KJ111802);重庆市卫生局医学科研项目(2012-1-096);重庆市高等教育教学改革研究重点项目(132128;133309);重庆万州区科技计划项目(201203055);重庆市应用开发计划项目(cstc2013yykf C10005)

摘　　要：目的构建大鼠CB1(r CB1)基因真核表达载体,检测r CB1基因在细胞中的表达,研究r CB1对人宫颈癌Ca Ski细胞凋亡的影响。方法从大鼠脑组织中提取总RNA,RT-PCR扩增r CB1基因,通过酶切、纯化、连接PCR纯化产物与p CDNA 3.1(+)质粒,构建pc DNA3.1(+)-r CB1。脂质体法将其转染到HEK293和Ca Ski细胞,Western blot及细胞免疫荧光联合激光扫描共聚焦方法检测r CB1的表达及定位,流式细胞仪检测细胞凋亡率,Western blot与实时荧光定量PCR检测r CB1、Bcl-2、Bax、Bad表达。结果酶切重组质粒获得5300 bp的载体片段和1500 bp的目的片段,测序结果和r CB1基因序列(NM_012784.4)一致。转染HEK293细胞后,r CB1在HEK293细胞细胞膜和细胞质表达。转染Ca Ski细胞后,r CB1使细胞凋亡率增加(P<0.05);r CB1基因上调Bax、Bad的表达,同时抑制Bcl-2的表达,与空白组比较,其差异具有显著性(P<0.05)。结论成功构建了pc DNA3.1(+)-r CB1真核表达载体,r CB1表达于细胞膜和细胞质,r CB1可以明显促进宫颈癌Ca Ski细胞凋亡,其机制是上调Bax、Bad和抑制Bcl-2表达。Objective To construct rCB1 gene eukaryotic expression vector, detect its expression in the cell, and explore its influence on apoptosis in human cervical cancer CaSki cells.Methods The total RNA was extracted from rat brains.The rCB1gene was amplified by RT-PCR.The pcDNA3.1（＋）-rCB1 was constructed by enzyme digestion, purifi-cation, bind the PCR purification products and pcDNA3.1 （＋） DNA.The pcDNA3.1 （＋）-rCB1 plasmid was transfect-ed into HEK293 and CaSki cells by liposomes.The expression and localization of rCB1 were detected by Western blot and immunofluorescence combined with confocal laser scanning microscopy.The apoptosis rate of CaSki cells was detected by flow cytometry.The expression of rCB1, Bcl-2, Bax and Bad was detected by Western blot and real-time fluorescence quantitative RT-PCR （qRT-PCR）.Results The 5300 bp pcDNA3.1（＋） and 1500 bp rCB1 were obtained after diges-ting the pcDNA3.1 （ ＋）-rCB1.The result of sequencing was in agreement with the sequence of rCB1 gene （ NM_012784.4 ） .The rCB1 expressed in the membrane and cytoplasm when pcDNA3.1 （＋）-rCB1 plasmid was transfected into HEK293 cells.The apoptosis rate of rCB1 group was increased compared with the blank group when pcDNA3.1 （＋）-rCB1 plasmid was transfected into CaSki cells （P〈0.05）.Compared with the blank group, rCB1 gene upregulated the expres-sion of Bax and Bad, and suppressed the expression of Bcl-2.The statistical difference was significant （ P 〈0.05）. Conclusions The pCDNA3.1（＋）-rCB1 eukaryotic expression vector is constructed successfully.It is found that rCB1 is expressed in membrane and cytoplasm of HEK293 cells.rCB1 can significantly promote the apoptosis in cervical cancer CaSki cells by up-regulating the expression of Bax and Bad, and down-regulating the expression of Bcl-2 as well.

关 键 词：CB1基因 HEK293细胞 CASKI细胞 细胞凋亡 rCB1 

分 类 号：Q95-33[生物学—动物学]